Although clinical use of aminoglycoside antibiotics is essential against life-threatening bacterial infections, there are serious ototoxic and nephrotoxic side-effects in 4-14 percent of all aminoglycoside prescriptions (4 million annually). Ototoxicity causes sensory hair cell death, hearing loss, and vestibular disorders, leading to physical, mental, educational, and language difficulties in patients. The overall aim of our studies is to identify, and then prevent the cellular mechanisms that initiate ototoxic drug-induced hair cell death. The long- term goal of this project is to develop interventional strategies that will allow future clinicians to use aminoglycosides without serious side effects. Gentamicin and forskolin are two unrelated ototoxic drugs that depolarize mitochondria, and increase production of reactive oxygen species and calcium levels in hair cells. Each of these toxic sequelae is a powerful trigger for inducing hair cell death. These common cellular responses to toxicity suggest that unrelated ototoxic drugs trigger a common pathway that lead to hair cell death. Therefore, we will use explants of bullfrog saccular hair cells to: (1) Identify the acute effects of gentamicin and forskolin in hair cells, and determine if they trigger these toxic sequelae by similar or differing mechanisms (2) Determine if inhibitors of drug-uptake can prevent toxic sequelae in hair cells (3) Determine if effective inhibitors of drug-uptake and toxic sequelae enhance hair cell survival during ototoxic drug treatment These studies provide a direct link between the molecular and biochemical studies of ototoxic drugs and the morphological analysis of fixed tissues after drug treatment. This knowledge will advance development of clinical strategies to prevent drug- induced hair cell death, and preserve inner ear function during this critical pharmaceutical therapy.